PROJECT SUMMARY Dietary changes in metal nutrients, including zinc and iron, influence the composition of the microbiota and correlate with increased infection susceptibility and gastrointestinal diseases, but the molecular mechanisms underlying these effects remain largely unknown. This lack of knowledge severely limits our ability to predict how diet or host metal status will impact treatment of gastrointestinal diseases or infection. Our long-term goal is to elucidate the molecular mechanisms governing how essential metals affect the human gut microbiota. The overall objective of the proposed work is to determine how essential metals affect growth and communication within probiotic bacterial communities of Lactobacillus species. Our research strategy is 1) to develop and apply protein-based fluorescent sensors that do not rely on oxygen and 2) to uncover molecular mechanisms through which metal ions affect gut microbiota homeostasis. Oxygen-insensitive protein-based fluorescent sensors will be used in live anaerobic cultures containing Lactobacillus to study metal uptake and how metal ion levels vary over time. Pure, multispecies, and in vitro gut model cultures will be used to evaluate how metal ion homeostasis varies with additional bacterial species and increasing complexity. Beyond direct detection and tracking of essential metals in culture with fluorescent sensors, we are carrying out systematic studies to measure how changes in essential metals affect Lactobacillus physiology and cell-cell communication (quorum sensing). Here, we are investigating the capacity of Lactobacillus species to store excess metal ions and aiming to identify the genes affected by varied metal levels in growth cultures. We are also measuring how varied metal levels affect the abundance of Lactobacillus quorum sensing signaling molecules. This research program is enhanced by collaborations with experts in microbiology, microbiome, and advanced fluorescence microscopy. The research is significant because it will provide mechanistic insight to how dietary metals affect gut microbiota composition and function. This insight is important because it will be useful for predicting the effects of metal-based dietary interventions and could potentially identify new targets to mitigate these effects. Furthermore, it will provide a knowledge basis for probiotic dietary interventions to combat gastrointestinal diseases and potentially identify new drug targets. The research is innovative because it represents a substantive departure from current work by shifting focus to uncover molecular-level mechanisms and roles for metal ions in gut bacteria that affect microbiota composition and function. By studying the Lactobacillus genus, we take advantage of well-established genetic approaches while focusing on an abundant organism in the small intestine, where most metal nutrient uptake occurs. Furthermore, Lactobacillus are well accepted as probiotics, but much remains to be learn...